One aspect of the current invention is an optimized nucleic acid delivery vehicle, or synthetic expression plasmid. The synthetic expression plasmid of this invention has reduced components, and has been optimized to increase efficacy, and reduce adverse reactions in vivo. In addition to a mammalian gene of interest, a typical nucleic acid delivery vehicle or synthetic expression plasmid contains many structural elements necessary for the in vitro amplification of the plasmid in a bacterial host. Consequently, some of the inherent bacterial nucleic acid sequences can cause adverse effects when the amplified plasmid is introduced into a mammalian host. For example, the presence of CpG sequences are known to cause both gene silencing and initiate an immune response in mammals. By utilizing codon optimization, essential bacterial structural elements (e.g. bacterial antibiotic resistant genes) are synthetically constructed and used to replace codons that contained detrimental sequences, but do not effect the final gene product. The current invention involves a “synthetic plasmid backbone” (pAV0201) that provides a clean lineage, which is useful for plasmid supplementation therapy in mammals.
A plasmid based mammalian expression system is minimally composed of a plasmid backbone, a synthetic delivery promoter in addition to the nucleic acid encoding a therapeutic expression product. A plasmid backbone typically contains a bacterial origin of replication, and a bacterial antibiotic selection gene, which are necessary for the specific growth of only the bacteria that are transformed with the proper plasmid. However, there are plasmids, called mini-circles, that lack both the antibiotic resistance gene and the origin of replication (Darquet et al., 1997; Darquet et al., 1999; Soubrier et al., 1999). The use of in vitro amplified expression plasmid DNA (i.e. non-viral expression systems) avoids the risks associated with viral vectors. The non-viral expression systems products generally have low toxicity due to the use of “species-specific” components for gene delivery, which minimizes the risks of immunogenicity generally associated with viral vectors. One aspect of the current invention is a new, versatile, and codon optimized plasmid based mammalian expression system that will reduce the adverse effects associated with prokaryotic nucleic acid sequences in mammalian hosts. In addition, this new plasmid will constitute the base of a species-specific library of plasmids for expression of hormones or other proteins for agricultural and companion animal applications.
Codon optimization: Expression of eukaryotic gene products in prokaryotes is sometimes limited by the presence of codons that are infrequently used in E. coli. Expression of such genes can be enhanced by systematic substitution of the endogenous codons with codons over represented in highly expressed prokaryotic genes. Although not wanting to be bound by theory, it is commonly thought that rare codons cause pausing of the ribosome. Pausing of the ribosome can lead to a failure to complete the nascent polypeptide chain and a uncoupling of transcription and translation. Additionally, pausing of the ribosome is thought to expose the 3′ end of the mRNA to cellular ribonucleases. An invention thought to circumvented such problems for prokaryotic expression of eukaryotic genes was discussed in U.S. Pat. No. 6,114,148 issued on Sep. 5, 2000 and titled “High level expression of proteins” with Seed, et al., listed as inventors (“the Seed '148 patent”). The Seed '148 patent features a synthetic gene that encodes a protein normally expressed in a mammalian cell wherein a non-preferred codon in the natural gene encoding the protein has been replaced by a preferred codon encoding the same amino acid. In contrast, the use of prokaryotic codons in mammalian systems can lead to detrimental effects (e.g. increased immune response). Furthermore, there are species specific differences with codons that are preferred, or less-preferred among species of a genus (Narum et al., 2001). One aspect of the current invention is the codon optimization of modified mammalian gene sequences. Publicly available databases for optimized codons have been referenced in the following articles: Nagata T, Uchijima M, Yoshida A, Kawashima M, Koide Y. Biochem Biophys Res Commun 261:445-51 (1999). Codon optimization effect on translational efficiency of DNA vaccine in mammalian cells: analysis of plasmid DNA encoding a CTL epitope derived from microorganisms; Uchijima, M, Yoshida, A, Nagata, T, Koide, Y. J Immunol 161:5594-9 (1998). Optimization of codon usage of plasmid DNA vaccine is required for the effective MHC class I-restricted T cell responses against an intracellular bacterium; Meetei, AR and Rao, MR. Protein Expr Purif 13:184-90 (1998). Hyperexpression of rat spermatidal protein TP2 in Escherichia coli by codon optimization and engineering the vector-encoded 5′ UTR; Andre, S, Seed, B, Eberle, J, Schraut, W, Bultmann, A, Haas, J. J Virol 72:1497-503 (1998). Increased immune response elicited by DNA vaccination with a synthetic gp120 sequence with optimized codon usage; Hale, R S and Thompson, G. Protein Expr Purif 12:185-8 (1998). Codon optimization of the gene encoding a domain from human type 1 neurofibromin protein results in a threefold improvement in expression level in Escherichia coli; Hubatsch, I, Ridderstrom, M, Mannervik, B. Biochem J 330:175-9 (1998). Human glutathione transferase A4-4: an alpha class enzyme with high catalytic efficiency in the conjugation of 4-hydroxynonenal and other genotoxic products of lipid peroxidation; Kim, C H, Oh, Y, Lee, T H. Gene 199:293-301 (1997). Codon optimization for high-level expression of human erythropoietin (EPO) in mammalian cells; Deng, T. FEBS Lett 409(2):269-72 (1997). Bacterial expression and purification of biologically active mouse c-Fos proteins by selective codon optimization; Cormack, B P, Bertram, G, Egerton, M, Gow, N A, Falkow, S, Brown, A J. Microbiology 143:303-11 (1997). Yeast-enhanced green fluorescent protein, a reporter of gene expression in Candida albicans; Prapunwattana, P, Sirawarapom, W, Yuthavong, Y, Santi, D V. Mol Biochem Parasitol 83:93-106 (1996) Chemical synthesis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene; Pikaart, M J and Felsenfeld, G. Protein Expr Purif 8:469-75 (1996). Expression and codon usage optimization of the erythroid-specific transcription factor cGATA-1 in baculoviral and bacterial systems; Yang, T T, Cheng, L, Kain, S R. Nucleic Acids Res 24:4592-3 (1996). Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein; Gouka, R J, Punt, P J, Hessing, J G, van den Hondel, C A. Appl Environ Microbiol 62:1951-7 (1996). Analysis of heterologous protein production in defined recombinant Aspergillus awamori strains; Altmann, S W, Timans, J C, Rock, F L, Bazan, J F, Kastelein, R A. Protein Expr Purif 6:722-6 (1995). Expression and purification of a synthetic human obese gene product; Kane, J., Current Opinion in Biotechnology 6:494-500 (1995). Effects of rare codon clusters on gene expression in Escherichia coli; Airenne, K J, Sarkkinen, P, Punnonen, E L, Kulomaa, M S. Gene, 144:75-80 (1994). Production of recombinant avidin in Escherichia coli; Wang, B Q, Lei, L, Burton, Z F. Protein Expr Purif 5:476-485 (1994). Importance of codon preference for production of human RAP74 and reconstitution of the RAP30/74 complex; Gerchman, S E, Graziano, V, Ramakrishnan, V. Protein Expr Purif 5:242-51 (1994). Expression of chicken linker histones in E. coli: sources of problems and methods for overcoming some of the difficulties; Dittrich, W, Williams, K L, Slade, M B. Bio/Technology 12:614-8 (1994). Production and Secretion of Recombinant Proteins in Dictyostelium discoideum; Holler, T P, Foltin, S K, Ye, Q Z, Hupe, D J. Gene 136:323-8 (1993). HIV1 integrase expressed in Escherichia coli from a synthetic gene; Kane, J F, Violand, B N, Curran, D F, Staten, N R, Duffin, K L, Bogosian, G. Nucleic Acids Res 20:6707-12 (1992). Novel in-frame two codon translational hop during synthesis of bovine placental lactogen in a recombinant strain of Escherichia coli; Kotula, Land Curtis, P J. Biotechnology (NY) 9:1386-9(1991). Evaluation of foreign gene codon optimization in yeast: expression of a mouse IG kappa chain; Makoff, A J, Oxer, M D, Romanos, M A, Fairweather, N F, Ballantine, S. Nucleic Acids Res. 17:10191-10202 (1989). Expression of tetanus toxin fragment C in E. coli: high level expression by removing rare codons; Misra, R and Reeves, P. Eur J Biochem 152:151-5 (1985). Intermediates in the synthesis of TolC protein include an incomplete peptide stalled at a rare Arg codon; Robinson, M, Lilley, R, Little, S, Emtage, J S, Yarranton, G, Stephens, P, Millican, A, Eaton, M, Humphreys, G. Nucleic Acids Res 12:6663-71 (1984). Codon usage can affect efficiency of translation of genes in Escherichia coli; Pedersen, S. EMBO J 3:2895-8 (1984). Escherichia coli ribosomes translate in vivo with variable rate.
As mentioned above, a plasmid backbone typically contains a bacterial origin of replication, and a bacterial antibiotic selection gene, which are necessary for the specific growth of only the bacteria that are transformed with the proper plasmid. However, the nucleotide sequence of the bacterial gene products can adversely affect a mammalian host receiving plasmid DNA. For example, it was desirable to avoid CpG sequences, as these sequences have been shown to cause a recipient host to have an immune response (Manders and Thomas, 2000; Scheule, 2000) to plasmids as well as possible gene silencing (Shi et al., 2002; Shiraishi et al., 2002). Thus, the DNA coding regions of any expressed genes avoid the “cg” sequence, without changing the amino acid sequence. Another aspect of the current invention involves the removal of unnecessary DNA sequences that were left over from prior cloning procedures. As a result of codon optimization, and removal of unnecessary DNA sequences, a synthetically generated plasmid backbone (“pAV0201”) with a unique cloning site that was constructed to generate a clean lineage of plasmid, which will be useful for plasmid mediated gene supplementation.
Growth Hormone (“GH”) and Immune Function: Another aspect of the current invention is utilizing the synthetically generated plasmid backbone pAV0201 for plasmid mediated gene supplementation. The central role of growth hormone (“GH”) is controlling somatic growth in humans and other vertebrates, and the physiologically relevant pathways regulating GH secretion from the pituitary is well known (Berneis and Keller, 1996). The GH production pathway is composed of a series of interdependent genes whose products are required for normal growth (Cuttler, 1996). The GH pathway genes include: (1) ligands, such as GH and insulin-like growth factor-I (“IGF-I”); (2) transcription factors such as prophet of pit 1, or prop 1, and pit 1: (3) agonists and antagonists, such as growth hormone releasing hormone (“GHRH”) and somatostatin (“SS”), respectively; and (4) receptors, such as GHRH receptor (“GHRH-R”) and the GH receptor (“GH-R”). These genes are expressed in different organs and tissues, including the hypothalamus, pituitary, liver, and bone. Effective and regulated expression of the GH pathway is essential for optimal linear growth, as well as homeostasis of carbohydrate, protein, and fat metabolism GH synthesis and secretion from the anterior pituitary is stimulated by GHRH and inhibited by somatostatin, both hypothalamic hormones (Frohman et al., 1992). GH increases production of IGF-I, primarily in the liver, and other target organs. IGF-I and GH, in turn, feedback on the hypothalamus and pituitary to inhibit GHRH and GH release. GH elicits both direct and indirect actions on peripheral tissues, the indirect effects being mediated mainly by IGF-I.
The immune function is modulated by IGF-I (Geffner, 1997; LeRoith et al., 1996), which has two major effects on B cell development: potentiation and maturation, and as a B-cell proliferation cofactor that works together with interlukin-7 (“IL-7”). These activities were identified through the use of anti-IGF-I antibodies, antisense sequences to IGF-I, and the use of recombinant IGF-I to substitute for the activity. There is evidence that macrophages are a rich source of IGF-I. The treatment of mice with recombinant IGF-I confirmed these observations as it increased the number of pre-B and mature B cells in bone marrow. The mature B cell remained sensitive to IGF-I as immunoglobulin production was also stimulated by IGF-I in vitro and in vivo.
The production of recombinant proteins in the last 2 decades provided a useful tool for the treatment of many diverse conditions. For example, GH-deficiencies in short stature children, anabolic agent in burn, sepsis, and AIDS patients (Carrel and Allen, 2000; Hart et al., 2001; Lal et al., 2000; Mulligan et al., 1999). However, resistance to GH action has been reported in malnutrition and infection (Kotzmann et al., 2001). Long-term studies on transgenic animals and in patients undergoing GH therapies have shown no correlation in between GH or IGF-I therapy and cancer development. GH replacement therapy is widely used clinically, with beneficial effects, but therapy is associated with several disadvantages (Blethen, 1995): GH must be administered subcutaneously or intramuscularly once a day to three times a week for months, or usually years; insulin resistance and impaired glucose tolerance (Burgert et al., 2002); accelerated bone epiphysis growth and closure in pediatric patients (Blethen and Rundle, 1996).
In contrast, essentially no side effects have been reported for recombinant GHRH therapies. Extracranially secreted GHRH, as mature peptide or truncated molecules (as seen with pancreatic islet cell tumors and variously located carcinoids) are often biologically active and can even produce acromegaly (Faglia et al., 1992; Melmed, 1991). Administration of recombinant GHRH to GH-deficient children or adult humans augments IGF-I levels, increases GH secretion proportionally to the GHRH dose, yet still invokes a response to bolus doses of recombinant GHRH (Bercu et al., 1997). Thus, GHRH administration represents a more physiological alternative of increasing subnormal GH and IGF-I levels (Corpas et al., 1993b).
GH is released in a distinctive pulsatile pattern that has profound importance for its biological activity. Secretion of GH is stimulated by the GHRH, and inhibited by somatostatin, and both hypothalamic hormones. GH pulses are a result of GHRH secretion that is associated with a diminution or withdrawal of somatostatin secretion. In addition, the pulse generator mechanism is timed by GH-negative feedback. The endogenous rhythm of GH secretion becomes entrained to the imposed rhythm of exogenous GH administration. Effective and regulated expression of the GH and insulin-like growth factor-I (“IGF-I”) pathway is essential for optimal linear growth, homeostasis of carbohydrate, protein, and fat metabolism, and for providing a positive nitrogen balance. Numerous studies in humans, sheep or pigs showed that continuous infusion with recombinant GHRH protein restores the normal GH pattern without desensitizing GHRH receptors or depleting GH supplies as this system is capable of feed-back regulation, which is abolished in the GH therapies. Although recombinant GHRH protein therapy entrains and stimulates normal cyclical GH secretion with virtually no side effects (Duck et al., 1992), the short half-life of GHRH in vivo requires frequent (one to three times a day) intravenous, subcutaneous or intranasal (requiring 300-fold higher dose) administration (Evans et al., 1985). Thus, as a chronic treatment, GHRH administration is not practical.
Wild type GHRH has a relatively short half-life in the circulatory system, both in humans and in farm animals (Frohman et al., 1986). After 60 minutes of incubation in plasma 95% of the GHRH(1-44)NH2 is degraded, while incubation of the shorter (1-40)OH form of the hormone, under similar conditions, shows only a 77% degradation of the peptide after 60 minutes of incubation (Frohman et al., 1989a). Incorporation of cDNA coding for a particular protease-resistant GHRH analog in a gene therapy vector results in a molecule with a longer half-life in serum (Draghia-Akli et al., 1999), increased potency, and provides greater GH release in plasmid-injected animals as described in in U.S. Pat. No. 6,551,996 that was issued on Apr. 23, 2003 titled “Super Active Porcine Growth Hormone Releasing Hormone Analog” with Schwartz, et al., listed as inventors, (“the Schwartz '996 patent”), the entire content is herein incorporated by reference. The Schwartz '996 patent teaches that an application of a GHRH analog containing mutations that improve the ability to elicit the release of growth hormone. In addition, the Schwartz '996 patent relates to the treatment of growth deficiencies; the improvement of growth performance; the stimulation of production of growth hormone in an animal at a greater level than that associated with normal growth; and the enhancement of growth utilizing the administration of growth hormone releasing hormone analog and is herein incorporated by reference. Mutagenesis via amino acid replacement of protease sensitive amino acids prolongs the serum half-life of the GHRH molecule. Furthermore, the enhancement of biological activity of GHRH is achieved by using super-active analogs that may increase its binding affinity to specific receptors as described in the Schwartz '996 patent.
Extracranially secreted GHRH, as processed protein species GHRH(1-40) hydroxy or GHRH(1-44) amide or even as shorter truncated molecules, are biological active. It has been reported that a low level of GHRH (100 pg/ml) in the blood supply stimulates GH secretion (Corpas et al., 1993a). Direct plasmid DNA gene transfer is currently the basis of many emerging therapeutic strategies and thus does not require viral genes or lipid particles (Aihara and Miyazaki, 1998; Lesbordes et al., 2002). Skeletal muscle is a target tissue because muscle fiber has a long life span and can be transduced by circular DNA plasmids that express over months or years in an immunocompetent host (Danko and Wolff, 1994; Wolff et al., 1992). Previous reports demonstrated that human GHRH cDNA could be delivered to muscle by an injectable myogenic expression vector in mice where it transiently stimulated GH secretion over a period of two weeks in immunocompetent mice (Draghia-Akli et al., 1997), and for 5 month in immunodeficient mice (Draghia-Akli et al., 2002)(human hormones are immunogenic in normal immunocompetent rodents, and transgene expression is transitory in these cases).
U.S. Pat. No. 5,061,690 issued on Oct. 29, 1991 and titled “Method for increasing milk production in mammals and/or increasing the birth weight of their newborn and improving postnatal growth “with Kann, et al., listed as inventors, (“the Kann '690 patent”). The Kann '690 patent is directed toward increasing both birth weight and milk production by supplying to pregnant female mammals an effective amount of human GHRH or one of it analogs for 10-20 days. Application of the analogs lasts only throughout the lactation period. However, multiple administrations are presented, and there is no teachings regarding administration of the growth hormone releasing hormone a nucleic acid delivery vehicle or a codon optimized synthetic mammalian expression plasmid.
U.S. Pat. No. 5,134,120 issued on Jul. 28, 1992 and titled “Use of growth hormone to enhance porcine weight gain” with Boyd, et al., listed as inventors, (“the Boyd '120 patent”); and U.S. Pat. No. 5,292,721 issued on Mar. 8, 1994 and titled “Use of growth hormone to enhance porcine fetal energy and sow lactation performance” with Boyd, et al., listed as inventors, (“the Boyd '721 patent”). Both the Boyd '120, and Boyd 721 patent teach that by deliberately increasing growth hormone in swine during the last 2 weeks of pregnancy through a 3 week lactation resulted in the newborn piglets having marked enhancement of the ability to maintain plasma concentrations of glucose and free fatty acids when fasted after birth. In addition, the Boyd '120 and Boyd '721 patents teach that treatment of the sow during lactation results in increased milk fat in the colostrum and an increased milk yield. These effects are important in enhancing survivability of newborn pigs and weight gain prior to weaning. However Boyd '120 and Boyd '721 patents provide no teachings regarding administration of the growth hormone releasing hormone a nucleic acid delivery vehicle or a codon optimized synthetic mammalian expression plasmid.
In summary, previous studies have shown that it is possible to treat various disease conditions in a limited capacity utilizing recombinant protein technology, but these treatments have some significant drawbacks. It has also been taught that nucleic acid expression plasmids that encode recombinant proteins are viable solutions to the problems of frequent injections and high cost of traditional recombinant therapy. However, the nucleic acid expression plasmids also have some drawbacks when injected into a mammalian host. The synthetic plasmids of this invention have reduced components, and have been codon optimized to increase efficacy, and reduce adverse reactions in vivo. The introduction of point mutations in to the encoded recombinant proteins was a significant step forward in producing proteins that are more stable in vivo than the wild type counterparts. Since there is a need in the art to expanded treatments for subjects with a disease by utilizing nucleic acid expression constructs that are delivered into a subject and express stable therapeutic proteins in vivo, the combination of codon optimization of an encoded therapeutic mammalian gene in an optimized plasmid backbone will further enhance the art of plasmid mediated gene supplementation.